Clopidogrel Synthesis Essay

1. Introduction

Arterial and venous thromboembolic disorders are associated with substantial morbidity and mortality. Acute coronary syndrome (ACS) is caused by thrombosis in the coronary arteries. Rupture of an atherosclerotic plaque triggers thrombogenesis by platelet activation and aggregation and activation of the coagulation cascade, leading to complete or partial vessel occlusion [1]. The current gold standard of care for short-term and long-term secondary prevention of cardiovascular events in patients with ACS is dual antiplatelet therapy with acetylsalicylic acid (ASA) and a thienopyridine such as clopidogrel [2,3]. Both ASA, an irreversible inhibitor of thromboxane A2 synthesis, and clopidogrel, an inhibitor of P2Y12 ADP platelet receptors, inhibit molecular pathways that mediate platelet activation and, therefore, prevent such adverse events [3]. However, despite the proven clinical benefit of these agents, patients remain at a substantial residual risk of recurrent cardiovascular events [4,5].

Arterial thrombosis involves both platelet aggregation and the activation of the coagulation cascade, providing the rationale for anticoagulant therapy in addition to antiplatelet therapy for secondary prevention of cardiovascular events in patients with ACS [1]. A number of studies have assessed the risks and benefits of warfarin therapy in addition to ASA [6,7] or dual antiplatelet therapy (ASA and clopidogrel) [8] for the prevention of cardiovascular events in patients with ACS. These studies showed an improvement in cardiovascular outcomes compared with antiplatelet therapy alone, but this improvement was accompanied by an increase in major bleeding. Currently, triple antithrombotic therapy with warfarin, ASA and clopidogrel is only recommended in patients at low risk of bleeding [8]. Warfarin is also associated with other limitations that often result in patients receiving inadequate prophylaxis or sub-optimal patient adherence. These limitations include multiple drug-drug and food-drug interactions and unpredictable responses that necessitate routine coagulation monitoring and dose adjustments to ensure that patients maintain an appropriate anticoagulation intensity [9].

Novel oral anticoagulants have been developed in recent years in an attempt to overcome some of the limitations associated with traditional agents (such as unfractionated heparin, low molecular weight heparins, fondaparinux and the vitamin K antagonists). These new agents, such as the direct Factor Xa inhibitors rivaroxaban and apixaban and the direct thrombin inhibitor dabigatran etexilate, have been investigated extensively in large-scale clinical trials across several indications, including ACS. Rivaroxaban has successfully completed a phase III clinical trial in patients with ACS [10]. The phase III trial of apixaban was terminated prematurely because of safety reasons [11] and a phase III trial for dabigatran etexilate in ACS has not been performed.

Rivaroxaban has shown a predictable pharmacokinetic/pharmacodynamic (PK/PD) profile, has a rapid onset of action, high oral bioavailability, few drug-drug interactions and does not require routine coagulation monitoring or dose adjustments for age, gender or body weight [12,13]. Rivaroxaban has been shown to be effective in animal models of arterial and venous thrombosis [13,14] and has demonstrated consistent efficacy and reassuring safety in large-scale clinical trials [10,15,16,17,18,19,20,21]. Rivaroxaban has gained approval for the prevention of venous thromboembolism after elective hip or knee replacement surgery in many countries worldwide. Rivaroxaban has also gained European and US approval for the prevention of stroke and systemic embolism in adult patients with non-valvular atrial fibrillation with one or more risk factors and European approval for the treatment of deep vein thrombosis (DVT) and prevention of recurrent DVT and pulmonary embolism following an acute DVT in adults.

A previous study in healthy subjects showed that ASA did not alter the PK/PD profile of rivaroxaban; these data also showed that the combination of rivaroxaban and ASA had no additional effect on platelet aggregation or bleeding time compared with ASA alone [22]. Clopidogrel is currently the most commonly used antiplatelet agent in patients with ACS, in clinical practice, and it is likely that some patients who receive rivaroxaban may also be treated with antiplatelet agents, such as clopidogrel [23].

The objectives of this study were to investigate the effect of co-administration of rivaroxaban and clopidogrel on bleeding time and platelet aggregation, and the potential influence of clopidogrel on the safety, tolerability, PD and PK of a single dose of 15 mg rivaroxaban, and vice versa, in healthy male subjects. Based on the mode of action and the characteristics of both study drugs, bleeding time was the only parameter that was expected to be affected by co-administration of rivaroxaban and clopidogrel in comparison with either drug alone.

2. Experimental Section

2.1. Subjects

This phase I study enrolled 27 healthy male subjects who were between 18 and 55 years of age, had a body mass index within the range of 18–32 kg/m2, a heart rate of 45–90 beats per minute, systolic blood pressure of 100–145 mmHg and diastolic blood pressure below 95 mmHg, and who had no relevant pathological changes in their electrocardiogram (ECG). Subjects were excluded if they had participated in any other clinical trial in the three months leading up to the study or had given more than 100 mL of blood in the previous four weeks or more than 500 mL of blood in the precedingthree months. Subjects were also excluded if they had any clinically relevant condition or medical history that may affect study results or if they had any medical condition that may affect their ability to participate or complete the study.

2.2. Study Design and Treatments

This randomized, non-blinded, single-centre, three-way crossover study was approved by the Ethics Committee of the North-Rhine Medical Council, Düsseldorf, Germany, and was conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, and German drug law. The study (study number 011864) was conducted at the Pharma Center of the Institute of Clinical Pharmacology, Bayer HealthCare AG, Wuppertal, Germany.

A clopidogrel response screening period preceded the study (Figure 1). During the screening period, subjects received a single dose of 300 mg clopidogrel and platelet aggregation was measured 24 h after administration. Fourteen of the 27 subjects showed more than 40% inhibition of platelet aggregation compared with baseline and were, therefore, considered to be clopidogrel responders. The initial dose of 300 mg clopidogrel was chosen for this study because this loading dose is generally used in patients with ACS [24]. A second dose of 75 mg clopidogrel was selected because this is the standard daily dose recommended by guidelines for use in patients with a variety of relevant conditions, including atrial fibrillation, stroke and ACS [24].

The 14 clopidogrel responders were randomly assigned to one of the following three treatments with a washout phase of about 14 days between treatments (Figure 1). Treatment A consisted of 300 mg clopidogrel on day 1 and 75 mg clopidogrel on day 2. Treatment B consisted of a single dose of 15 mg rivaroxaban. Treatment C combined treatments A and B, with rivaroxaban given on day 2. Subjects were hospitalized on the evening before treatment was started in the morning and they stayed on the study ward for 3 days (treatment A), 2 days (treatment B) and 5 days (treatment C) after the first drug dose, and were discharged thereafter if there were no medical objections. The study ended with the final assessments approximately 1 week after the last treatment. All treatments were administered after 10 h of fasting at 08:00. Lunch was scheduled 4 h after tablet intake.

Rivaroxaban, at doses between 5 mg and 80 mg, has been shown to have relevant PD effects [25,26,27]. Based on these results, a dose of 15 mg rivaroxaban was chosen as a suitable dose for this study. More recently, daily doses of 5–20 mg have been shown to be clinically efficacious in phase II and phase III trials [15,16,17,18,19,20,21,27,28].

2.3. Safety and Tolerability

Safety and tolerability were assessed subjectively and objectively. Subjective assessment was obtained by asking the subjects non-leading questions about the occurrence of any adverse events or by spontaneous reporting of adverse events. Adverse events were classified according to their degree of severity. Objective tolerability was evaluated by monitoring cardiovascular parameters including heart rate, blood pressure and ECG parameters. In addition, blood tests, clinical chemistry, urine test and drug screening were part of the objective assessment.

Figure 1. Study design of the three-way crossover study. * Treatment period during which one subject withdrew from the study owing to an adverse event.

Figure 1. Study design of the three-way crossover study. * Treatment period during which one subject withdrew from the study owing to an adverse event.

2.4. Bleeding Time

Bleeding time tests were performed in accordance with the protocol reported by the International Committee on Standardization of the Bleeding Time [29]. Bleeding time measurements depend on the method and on who carries out the procedure; therefore, results vary greatly between studies. To reduce variability, one study nurse was specifically trained in the procedure of the test before this study. The same study nurse carried out all measurements at all time points for each subject. Briefly, the bleeding time test was performed on the lateral aspect of the volar surface of the forearm 3–5 cm distal to the elbow crease in an area devoid of hair, scars, bruises or surface veins. A sphygmomanometer on the upper arm was inflated to 40 mmHg for 30 seconds before the incision was made and maintained at 40 mmHg until the end of the procedure. The bleeding time incision was made by placing a Surgicutt Adult® (ITC, Edison, NJ, USA) device (used in accordance with the recommendations of the manufacturer, as specified in the package insert) gently against the forearm, perpendicular (vertical) to the elbow crease. After the incision, the drops of blood flowing from the wound were wicked with filter paper (Rundfilter by Schleich und Schuell, diameter 70 mm) every 30 seconds until bleeding ceased, with care taken not to touch the incision or dislodge the developing platelet plug. The bleeding time was measured in seconds as the total duration of blood flow from the wound. A closure was placed across the incision, and the subjects were asked not to remove it for 24 h.

2.5. Pharmacodynamic Parameters

The effects of rivaroxaban and clopidogrel on Factor Xa activity, prothrombin time (PT), activated partial thromboplastin time (aPTT) and HepTest were assessed, as described previously [26]. Factor Xa activities above 0.1 IU/mL (the lower limit of quantification) were determined with a precision of 3.8–6.6% and an accuracy of 96–114%. PT (assessed using freeze-dried thromboplastin from rabbit brain (Neoplastine Plus; Roche Diagnostics, Mannheim, Germany), aPTT [assessed using a kaolin-activated test (Roche Diagnostics)], and HepTest® (Haemachem, St. Louis, MO, USA) were measured with a ball coagulometer (KC 10, Amelung, Germany) in accordance with the manufacturer’s instructions. Blood samples were taken at the time of administration of rivaroxaban and again after 0.5, 1, 2, 3, 4, 6, 8, 12, 15, 24 and 48 h (treatment B). When clopidogrel and rivaroxaban were co-administered on day 2 of treatment C, blood samples were collected at the same time points and after 96 h. When only clopidogrel was administered (treatment A), blood samples were taken after 4, 8, 24 and 48 h. Blood samples were centrifuged to obtain plasma samples, which were then frozen and stored below −15 °C until analysis.

2.5.1. Platelet Aggregation

Platelet aggregation was determined by the Born method just before and 4 h after study drug administration [30]. This method is based on the turbidimetric determination of cell suspensions. Because platelet-enriched plasma possesses a minor permeability for long-wave light (whereas plasma poor in platelets lets light pass through without obstruction), the percentage of light permeability of plasma can be used as a measure for platelet aggregation. Measurement was performed at 650 nm. The individual response of changes of platelet aggregation to baseline was determined using 29 µM ADP in addition to an optimized individual activating regimen using ADP. The light transmission of each aggregated sample is reported in relation to the light transmission of the individual platelet-enriched plasma sample (0% light transmission) and the corresponding individual platelet-poor plasma sample (prepared by centrifugation; 100% light transmission). Within 5 minutes, the maximum aggregation and the maximum gradient were evaluated against the different ADP concentrations that were used for stimulation.

2.5.2. Platelet Activation Markers

The relative changes to baseline at 4 h after drug administration of the platelet activation markers membrane glycoprotein receptor GPIIb/IIIa, P-selectin and annexin V were determined by flow cytometry [31]. To assure uniformity of assays and to minimize sample manipulation, blood samples were mixed gently and processed without delay. Platelet-rich plasma was prepared by slow centrifugation, and platelets were labelled with monoclonal antibodies against GPIIb/IIIa (CD41a), P­selectin (CD62p) and annexin V protein. Samples were analysed using a COULTER® EPICS® XL™ flow cytometer (Beckman Coulter, Inc.; Brea, CA, USA). The average immunofluorescence of the total population or the fraction of activated events was determined and compared with individual control samples before treatment.

2.6. Pharmacokinetic Parameters

Blood samples for analyses of the PK parameters of rivaroxaban (area under the concentration-time curve [AUC], maximum plasma concentration [Cmax], terminal half-life [t½] were taken at the time of administration of rivaroxaban and again after 0.5, 1, 2, 3, 4, 6, 8, 12, 15, 24 and 48 h for treatment B and treatment C). PK parameters were determined by non-compartmental analysis using KINCALC® (Bayer HealthCare AG, Wuppertal, Germany). Quantitative analysis of rivaroxaban in plasma was performed using a fully validated assay. Briefly, concentrations of rivaroxaban were determined after solid/liquid extraction by high-performance liquid chromatography coupled with a tandem mass spectrometer. A close chemical analogue of rivaroxaban, BAY 60-4758 (5-chloro-N-({3­[3,5-dimethyl-4-(3-oxomorpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide), was used as the internal standard. Prior to the high-performance liquid chromatography analysis, rivaroxaban and the internal standard were extracted from the matrix by solid phase extraction using C18 cartridges. The calibration range of the procedure was from 0.5 µg/L (lower limit of quantification) to 500 µg/L. Quality control samples in the concentration range from 1.35 µg/L to 266 µg/L were determined with an accuracy of 96.3–98.2% and a precision of 3.45–7.91% (n = 15 each).

2.7. Statistical Analysis

Statistical evaluations were performed with the use of the SAS® software package version 8.2 at the Department of Global Pharmacometrics (Bayer HealthCare AG, Wuppertal, Germany). Bleeding time results and platelet aggregation results were analysed for each subject 4 h after drug administration using descriptive statistical methods. Student’s paired t tests were used to compare treatments for these parameters, and 90% confidence intervals (CIs) for the difference were calculated.

The primary PK parameters AUC and Cmax of rivaroxaban were analysed assuming log-normally distributed data. The logarithms of AUC and Cmax were analysed by analysis of variance (ANOVA) including sequence, subject (sequence), period and treatment effects. Based on these analyses, a point estimator (least squares-means) and 90% CI for the ratio (rivaroxaban + clopidogrel)/(rivaroxaban alone) was calculated by re-transformation of the logarithmic data using the intraindividual standard deviation of the ANOVA.

Sanofi Synthesis of (±) Clopidogrel

 

 

 

RPG Synthesis of (±) Clopidogrel

 

 

 

It is interesting to note that only the enantiomerically pure (+)-clopidogrel (dextrorotatory, rotates plane polarised light in a clockwise direction) exhibits platelet aggregation inhibiting activity while the (-)- clopidogrel (levorotatory, rotates plane polarised light in an anticlockwise direction) is inactive. Enantiomerically pure (+) clopidogrel used to be obtained from resolution of the racemic clopidogrel. If racemic clopidogrel is mixed with levorotatory camphor-10-sulfonic acid in acetone then a salt is obtained which can be recrystallised from acetone to generate (+)-clopidogrel

 

 

Asymmetric synthesis of (+)- clopidogrel

 

                                

 

Syntheses taken from J.Li, et al, Contemporary Drug Synthesis, Wiley Interscience, 2004.

 

 

Comments

Leave a Reply

Your email address will not be published. Required fields are marked *